Isolation of nucleic acids

ABSTRACT

Provided herein is technology relating to isolating nucleic acids. In particular, the technology relates to methods and kits for extracting nucleic acids from problematic samples such as stool.

The present application claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 61/485,214, 61/485,338, 61/485,386, and61/485,448, each of which was filed May 12, 2011, and each of which isincorporated herein by reference in its entirety.

FIELD OF INVENTION

Provided herein is technology relating to isolating nucleic acids. Inparticular, the technology relates to methods and kits for extractingnucleic acids from problematic samples such as stool.

BACKGROUND

Isolating specific target nucleic acids from a sample is an importantstep for many medical diagnostic assays. For example, certain mutationsand methylation states in known genes are correlated, associated, and/orpredictive of disease. DNA harboring these genes can be recovered from asample and tested for the presence of the particular mutations andmethylation states.

In practice, such assays require isolating and assaying several genetictargets from a sample. For many detection methods, detecting raremutations or methylation events in a single gene requires isolating andtesting a large quantity of DNA. This problem is compounded whenassaying a panel of genes, each of which must be present in a largequantity for a robust diagnostic test. Thus, to detect rare mutationsand methylation events in multiple genes, the isolated DNA must behighly concentrated and comprise a substantial portion of the detectionassay.

This requirement imposes many problems, however. For example, preparingsuch quantities and concentrations of DNA requires a large sample asinput (e.g., having a mass of several grams, e.g., approximately 2-4grams) to provide sufficient nucleic acid for detection, and thusrequires a method that can prepare DNA from a large sample. In addition,assay inhibitors are often isolated and concentrated with the DNApreparation. Consequently, concentrated DNA preparations produced byconventional methods also often retain unacceptable concentrations ofinhibitors, which are then introduced into a subsequent assay. Moreover,if all targets of the panel are extracted simultaneously in a bulk,non-selective DNA preparation, the sensitivity of the assay iscompromised because, as the preparation is divided into aliquots fortesting, less extracted DNA from any one gene of the panel is present inthe assay. If, on the other hand, all members of the panel are extractedand tested together and are thus present in the same assay mixture, thesensitivity of detecting any single particular target is compromised bythe presence of the non-target DNA molecules.

In addition, if a particular diagnostic target is present in a complexsample, it will be present in a small amount relative to othermaterials—both nucleic acid and non-nucleic acid—in the sample, thusproviding a challenge for analytical methods designed to detect it. Forexample, analyses of DNA from stool samples is complicated by the factthat bacteria compose approximately 60% of the dry mass of feces and theremainder is largely the remains of plant and animal matter ingested asfood by the subject. As such, the human subject's cells, which are onlythose that slough off the lining of the digestive tract, are a verysmall fraction of the stool and substantial amounts of nucleic acidsfrom other sources are present. Furthermore, in assays to detect genemodifications indicative of colon cancer, cells derived from a tumorthat may be present in the colon would compose only a small fraction ofthe human subject's gut cells that slough off the digestive tractlining. Consequently, cancer cells (and the DNAs they contain) make up aminimal amount of the stool mass. Such samples are also often veryviscous, which presents problems in sample preparation and isolation ofnucleic acid.

Conventional methods and kits for isolating DNA from samples typicallyprepare total DNA (e.g., by a non-specific precipitation method) from asample. For complex samples such as stool samples, this is a particulardrawback of conventional methods, as total DNA isolated from a stoolsample comprises DNA from the gut-resident bacteria (and any viruses,eukaryotes, and archaea present) along with DNA from the subject.Moreover, conventional methods and kits are primarily designed toprepare DNA from small samples, e.g., samples having masses of less than1 gram, e.g., 50 to 200 milligrams, limiting the yield of target nucleicacid from complex samples to very small amounts. Additional drawbacksare that most conventional technology does not effectively removeinhibitors and often require long processing steps, e.g., incubations.Consequently, conventional methods are not suited to high-sensitivityand high-specificity multi-gene panel analysis because they cannotprepare sufficient amounts of highly concentrated, inhibitor-free DNAfrom large samples, such as a stool sample of several grams. Assaysusing DNA prepared with conventional methods will not provide a samplethat can be assayed with the required sensitivity threshold fordetecting rare mutation or methylation events. Using a conventionalmethod or kit to attain the starting quantities needed to attain suchsensitivity requires multiple DNA extractions (e.g., the use of multiplekits) from multiple samples in addition to extra purification steps toremove inhibitors. Therefore, what is needed is a method of preparingconcentrated, inhibitor-free DNA from a sample for each member of a genepanel for use in diagnostic assays.

SUMMARY

Provided herein is technology relating to isolating nucleic acids. Inparticular, the technology relates to methods, systems, and kits forextracting and purifying nucleic acids from exfoliated intestinal cellsin stool specimens for use in quantitative and sensitive assays. Thetechnology is embodied in a novel method for purifying specific DNA fromstool that utilizes inhibitor removal steps and direct capture of DNAfrom stool supernatant, or a combination of these steps. The technologyfurther provides filtration devices suitable for use with complex andviscous samples, such as stool samples. Accordingly, provided herein isa method for isolating a target nucleic acid from a sample, the methodcomprising removing an assay inhibitor, if present, from the sample toproduce a clarified sample; capturing the target nucleic acid, ifpresent, from the clarified sample with a capture reagent to form acapture complex; isolating the capture complex from the clarifiedsample; and recovering the target nucleic acid, if present, from thecapture complex in a nucleic acid solution. In some embodiments themethod further comprises retaining the clarified sample after thecapturing step; and repeating the isolating and recovering steps usingthe retained clarified sample and a second capture reagent.

In some embodiments, removing the inhibitor comprises homogenizing thesample to produce a homogenate; centrifuging the homogenate to produce asupernatant; treating the supernatant with an inhibitor-adsorbingcomposition to bind the inhibitor, if present, in an inhibitor complex;and isolating the inhibitor complex from the supernatant to produce aclarified sample. The inhibitor-adsorbing composition in someembodiments is a polyvinylpyrrolidone. In some embodiments, thepolyvinylpyrrolidone is insoluble and in some embodiments thepolyvinylpyrrolidone is a polyvinylpolypyrrolidone. It is useful in someembodiments to provide the polyvinylpyrrolidone in a premeasured form,for example in some embodiments the polyvinylpyrrolidone is provided asa tablet. Various techniques are used to separate the inhibitor complexfrom the sample. For example, in some embodiments isolating theinhibitor complex comprises centrifuging to separate the inhibitorcomplex from the supernatant.

In some embodiments, the centrifuging comprises centrifuging through aspin column. Therefore, in some embodiments provided herein istechnology relating to filtration and particularly, but not exclusively,to filters and methods for filtering by means of centrifugation.Specifically, some embodiments of the technology provided herein addressthe problem of spin filter clogging by providing technology in whichboth the bottom end and body of a spin filter are made from a porous orpermeable material. That is, the walls of the spin filter are made ofthe same or similar material as that used for the filter means at thebottom end in conventional designs. As such, when the bottom portion ofthe filter becomes clogged during filtration, the walls provideadditional surface through which the sample can be filtered.

This technology is provided herein as a spin filter comprising a hollowbody, a bottom end, and an open top end opposite the bottom end, whereinthe hollow body is made from a porous filtering material. In someembodiments the bottom end is made from a porous filtering material. Thehollow body and bottom end of the spin filter assume any shapeappropriate for the filtration application to which the filter isapplied. For example, in some embodiments the hollow body is a tube andin some embodiments the bottom end is a hemisphere. In otherembodiments, the bottom end is a disc, a cone, or a portion of anellipsoid. Furthermore, the spin filter is made from any material thatis appropriate for filtering a sample. Thus, in some embodiments theporous filtering material is polyethylene. Samples comprise varyingsizes of particles, matter, precipitates, etc. that are to be removed byfiltration. Accordingly, the filtering material can be selected to havephysical properties that provide the desired separation. For example, insome embodiments the porous filtering material has a nominal pore sizeof 20 micrometers. In some embodiments, use of the filter produces afiltrate that a user retains for additional processing. As such, someembodiments provide a spin filter assembly comprising a spin filter asdescribed and a collection vessel adapted to receive the spin filter andcollect the filtrate.

Also provided herein are methods for producing a filtrate from a samplecomprising placing a sample to be filtered into the spin filter andcentrifuging the spin filter, wherein during centrifuging, a fraction ofthe sample passes through porous filtering material of said spin filterto produce a filtrate.

The technology can be provided as a kit for use in a sample separation.Embodiments of such a kit comprise a spin filter as described and aninstruction for use. In some embodiments the kit further comprises acollection vessel. In some embodiments, a kit comprising a spin filterfurther comprises additional reagents and materials for samplepreparation, e.g., for inhibitor removal and/or target nucleic acidisolation.

In some embodiments, the methods and systems of the technology comprisecapturing a nucleic acid target. Capturing the target nucleic acid, insome embodiments, comprises exposing a sample, such as a clarifiedsample preparation, to a denaturing condition to produce a denaturedsample; and binding target nucleic acid in the denatured sample to acapture reagent to form a capture complex. Many treatments andconditions find use in denaturing macromolecules such as DNA. Forexample, in some embodiments, the denaturing condition comprisesheating, e.g., in some embodiments the denaturing condition comprisesheating at 90° C. Supplementing the sample to be denatured facilitatesthe denaturing; accordingly, in some embodiments, the clarified samplefurther comprises a denaturant. In certain preferred embodiments, thedenaturant comprises guanidine thiocyanate. Furthermore, in someembodiments the capture reagent comprises an oligonucleotidecomplementary to at least a portion of the target nucleic acid. In somepreferred embodiments, the capture reagent comprises particle, e.g., amagnetic particle. The oligonucleotide, in some embodiments of thetechnology, hybridizes to at least a portion of the target nucleic acid,and thus in some embodiments, the binding step comprises hybridizing theoligonucleotide and the target nucleic acid. Isolating the capturereagent (e.g., the capture reagent/target nucleic acid complex) isaccomplished in some embodiments by exposing the capture reagent to amagnetic field; that is, in some embodiments provided herein, theisolating step comprises exposing the capture complex to a magneticfield and in some embodiments exposing the capture complex to themagnetic field localizes the target nucleic acid. The magnetic field isproduced by any appropriate magnet or magnetic device for the method.For example, in some embodiments the isolating step comprises placingthe sample in a magnetic field produced by a first magnet oriented withits north pole in close proximity to the sample and a second magnetoriented with its south pole in close proximity to the sample; andwaiting for a time sufficient to allow the magnetic field to move themagnetic particles to the desired location. A device for producing astrong magnetic field is described, for example, in U.S. patentapplication Ser. No. 13/089,116, incorporated by reference herein.

The technologies provide for recovering target nucleic acid from thecapture reagent. In some embodiments, recovering the target nucleic acidcomprises eluting the target nucleic acid from the capture complex,e.g., in some embodiments, by heating. In some embodiments, elution ofthe target nucleic acid from the capture complex comprises exposing thecapture complex to high pH, e.g., in some embodiments, by adding asolution of sodium hydroxide.

In some embodiments, the technology provides methods, systems and kitsfor capturing multiple nucleic acids from a single sample, e.g., a stoolsample. For example, provided herein are methods for isolating a nucleicacid from a stool sample comprising contacting a stool sample with atarget-specific capture reagent; binding a target nucleic acid, whenpresent, to the target-specific capture reagent to form a complex;isolating the complex comprising the target-specific capture reagent andthe target nucleic acid, when present, from the stool sample; elutingthe target nucleic acid, when present, from the complex to produce atarget nucleic acid solution comprising the target nucleic acid, whenpresent; and repeating the method using a different target-specificcapture reagent. The methods are appropriate for large samples, e.g.,having a mass of at least 4 grams. Moreover, each eluted target nucleicacid is sufficiently purified, sufficiently concentrated, andsufficiently free of inhibitors such that each eluted target nucleicacid, when present, is detected by a quantitative PCR when the targetnucleic acid solution composes up to approximately one-third of a volumeof the quantitative PCR.

In some embodiments of the methods provided, the target nucleic acid isa human target nucleic acid. In additional embodiments, the targetnucleic acid is a DNA. While not limited in the means by which thenucleic acid is isolated from the stool sample, in some embodiments thetarget-specific capture reagent is a sequence-specific nucleic acidcapture reagent. In some embodiments, the sequence-specific nucleic acidcapture reagent is an oligonucleotide and in some embodiments theoligonucleotide is covalently attached to a magnetic or paramagneticparticle. Some embodiments provide that a magnet is used for theisolating step and some embodiments provide for the simultaneousisolation of more than one target using multiple target-specific capturereagents in a single isolation step.

The method is not limited in the types of samples that are processed.For example, in some embodiments the sample is a viscous sample, e.g.,having a viscosity of more than ten centipoise in some embodiments andhaving a viscosity of more than twenty centipoise in some embodiments.Additionally, the samples are of a wide range of sizes. The methods areused to process samples having, in some embodiments, a mass of more thanone gram and in some embodiments the sample has a mass of more than fivegrams.

The technology provided herein is directed to removing inhibitors fromsamples below an amount that inhibits an assay. Thus, in someembodiments, the method provides that the nucleic acid solutioncomprises a first amount of the assay inhibitor that is less than asecond amount of the assay inhibitor, wherein the second amount of theassay inhibitor inhibits PCR when five microliters of the nucleic acidsolution are used in a PCR having a volume of twenty-five microliters.In some embodiments, the nucleic acid solution comprises a first amountof the assay inhibitor that is less than a second amount of the assayinhibitor, wherein the second amount of the assay inhibitor inhibits PCRwhen one microliter of the nucleic acid solution is used in a PCR havinga volume of twenty-five microliters.

The technology is related to medical molecular diagnostics whereinquerying the state, presence, amount, sequence, etc., of a biologicalsubstance (e.g., a molecule) is used to aid a medical assessment.Accordingly, in some embodiments, the target nucleic acid is correlatedwith a disease state selected from the set consisting of colon cancerand adenoma.

The technology described herein is provided in a kit form in someembodiments—for example, embodiments provide that the technology is akit for isolating a target nucleic acid from a sample comprising acapture reagent comprising an oligonucleotide covalently attached to amagnetic particle, an apparatus to produce a magnetic field,polyvinylpyrrolidone, and an instruction for use. In some embodiments,the kit further comprises a homogenization solution. In someembodiments, the kit further comprises an elution solution and in someembodiments the kit further comprises guanidine thiocyanate. In someembodiments, it is convenient for the polyvinylpyrrolidone to be in apremeasured form. For example, the polyvinylpyrrolidone is provided in atablet or capsule in some embodiments. Some embodiments of the kitprovide a spin filter for removing polyvinylpyrrolidone.

In some embodiments, the target nucleic acid is isolated using amagnetic field. As such, embodiments of the kits described hereinprovide an apparatus that produces a magnetic field. One device that isused to produce a magnetic field suitable for use with embodiments ofthe technology provided herein comprises two magnets or sets of magnetsand places the north pole(s) of the first magnet or set of magnets inclose proximity to the sample and the south pole(s) of the second magnetor set of magnets in close proximity to the sample. In some embodiments,the kits further provide a device for collecting a sample, e.g., adevice having a body and a detachable sample capsule attached to thebody, wherein the detachable sample capsule comprises a samplecollection space adapted to enclose a sample (for example, as describedin U.S. Pat. Appl. Ser. No. 61/476,707).

In some embodiments, the kit provides vessels (e.g., a tube, a vial, ajar, and the like) used to process samples and hold various compositionsused to process samples or that result from processing samples. Forexample, in some embodiments the kit further comprises a vessel in whichto hold the sample and in some embodiments the kit further comprises avessel in which to hold the isolated target nucleic acid. The kit, insome embodiments, is used at a location other than where the sample isprocessed and/or where the analyte is assayed. Accordingly, in someembodiments the kit further comprises a shipping container.

The technology provided herein finds use in systems for preparing anucleic acid from a sample. In some embodiments, the system comprisespolyvinylpyrrolidone for removing an inhibitor from the sample, areagent for capturing a target nucleic acid from the sample, and afunctionality for producing a magnetic field. In some embodiments, thesystem further comprises a functionality for collecting the sample andin some embodiments the system further comprises a functionality forshipping the nucleic acid solution.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIGS. 1A and 1B provide charts of aspects of the nucleic acid isolationprocess. FIG. 1A provides a chart showing the steps of the nucleic acidisolation process. FIG. 1B is a flowchart showing an embodiment of theprocess that finds use in the sequential extraction of multiple targetsfrom the same sample as an aspect of the overall process of FIG. 1A.

FIG. 2 is a chemical structure of a polyvinylpyrrolidone.

FIG. 3 is a drawing of an exemplary spin filter.

FIG. 4 is a drawing showing an exploded view of the spin filter shown inFIG. 3.

FIGS. 5A-5C are a series of drawings showing spin filter bottom endsassociated with the spin filter of FIGS. 3 and 4. FIG. 5A is a drawingof a disc-shaped, solid (e.g., non-porous or non-permeable) bottom end;FIG. 5B is a drawing of a disc-shaped, porous (permeable) bottom end;FIG. 5C is a drawing of a porous, conical bottom end.

FIG. 6 is a drawing of a spin filter assembled with a collection tube.

FIG. 7 is a cut-away drawing of the spin filter depicted in FIG. 6.

FIGS. 8A and 8B are drawings of a spin filter comprising a body of aporous material and a bottom end provided by a filter support. FIG. 8Ais an assembled view and FIG. 8B is an exploded view.

FIGS. 9A-9D are plots showing the removal of inhibitors from a stoolsample.

FIGS. 10A-10D are plots showing that spin filtration improves theremoval of inhibitors.

FIG. 11A is a plot of data comparing the localization efficiency of theconventional technology for samples having viscosities of 1 centipoiseand 25 centipoise. FIG. 11B is a plot of data comparing the localizationefficiency of the magnetic localization device provided by Light andMiller (U.S. patent application Ser. No. 13/089,116) provided forsamples having viscosities of 1 centipoise and 25 centipoise.

FIG. 12A is a plot showing the results of a quantitative PCR in which asingle extraction from a stool sample recovers most of the target DNA.FIG. 12B shows the concentrations of Gene A and Gene V in nucleic acidsolutions from a first extraction and a second extraction.

FIGS. 13A-13D show plots showing the results of quantitative PCRs inwhich the recoveries of four target DNAs are similar regardless of theorder in which the four target DNAs are extracted from a stool sample.

FIG. 14 provides a chart comparing the workflow of an embodiment(Process A) with an exemplary process for isolating DNA from stoolsamples using steps based on existing methods (Process B, see, e.g., WO2010/028382).

DETAILED DESCRIPTION

The present technology is related to producing DNA samples and, inparticular, to methods for producing DNA samples that comprise highlypurified, low-abundance nucleic acids in a small volume (e.g., less than100, less than 60 microliters) and that are substantially and/oreffectively free of substances that inhibit assays used to test the DNAsamples (e.g., PCR, INVADER, QuARTS, etc.). Such DNA samples find use indiagnostic assays that qualitatively detect the presence of, orquantitatively measure the activity, expression, or amount of, a gene, agene variant (e.g., an allele), or a gene modification (e.g.,methylation) present in a sample taken from a patient. For example, somecancers are correlated with the presence of particular mutant alleles orparticular methylation states, and thus detecting and/or quantifyingsuch mutant alleles or methylation states has predictive value in thediagnosis and treatment of cancer.

Many valuable genetic markers are present in extremely low amounts insamples and many of the events that produce such markers are rare.Consequently, even sensitive detection methods such as PCR require alarge amount of DNA to provide enough of a low-abundance target to meetor supersede the detection threshold of the assay. Moreover, thepresence of even low amounts of inhibitory substances compromise theaccuracy and precision of these assays directed to detecting such lowamounts of a target. Accordingly, provided herein are methods providingthe requisite management of volume and concentration to produce such DNAsamples.

Some biological samples, such as stool samples, contain a wide varietyof different compounds that are inhibitory to PCR. Thus, the DNAextraction procedures include methods to remove and/or inactivate PCRinhibitors. As such, provided herein is technology relating toprocessing and preparing samples and particularly, but not exclusively,to methods, systems, and kits for removing assay inhibitors from samplescomprising nucleic acids.

DEFINITIONS

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

As used herein, “a” or “an” or “the” can mean one or more than one. Forexample, “a” widget can mean one widget or a plurality of widgets.

As used herein, an “inhibitor” means any compound, substance, orcomposition, or combination thereof, that acts to decrease the activity,precision, or accuracy of an assay, either directly or indirectly, withrespect to the activity, precision, or accuracy of the assay when theinhibitor is absent. An inhibitor can be a molecule, an atom, or acombination of molecules or atoms without limitation.

As used herein, the process of passing a mixture through a filter iscalled “filtration”. The liquid produced after filtering a suspension ofa solid in a liquid is called “filtrate”, while the solid remaining inthe filter is called “retentate”, “residue”, or “filtrand”.

As used herein, “insoluble” refers to the property that a substance doesnot substantially dissolve in water and is essentially immiscibletherewith. Upon separation of an aqueous phase from a non-aqueous phase,an insoluble substance does not partition into or partition with theaqueous phase.

As used herein, the terms “subject” and “patient” refer to any animal,such as a dog, cat, bird, livestock, and particularly a mammal,preferably a human. In some instances, the subject is also a “user” (andthus the user is also the subject or patient).

As used herein, the term “sample” and “specimen” are usedinterchangeably, and in the broadest senses. In one sense, sample ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from animals (including humans) and encompass fluids, solids,tissues, and gases. Biological samples include blood products, such asplasma, serum, stool, urine, and the like. Environmental samples includeenvironmental material such as surface matter, soil, mud, sludge,biofilms, water, crystals, and industrial samples. Such examples are nothowever to be construed as limiting the sample types applicable to thepresent invention.

The term “target,” when used in reference to a nucleic acid capture,detection, or analysis method, generally refers to a nucleic acid havinga feature, e.g., a particular sequence of nucleotides to be detected oranalyzed, e.g., in a sample suspected of containing the target nucleicacid. In some embodiments, a target is a nucleic acid having aparticular sequence for which it is desirable to determine a methylationstatus. When used in reference to the polymerase chain reaction,“target” generally refers to the region of nucleic acid bounded by theprimers used for polymerase chain reaction. Thus, the “target” is soughtto be sorted out from other nucleic acid sequences that may be presentin a sample. A “segment” is defined as a region of nucleic acid withinthe target sequence. The term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of a target.

As used herein, the term “locus” refers to a particular position, e.g.,of a mutation, polymorphism, or a C residue in a CpG dinucleotide,within a defined region or segment of nucleic acid, such as a gene orany other characterized sequence on a chromosome or RNA molecule. Alocus is not limited to any particular size or length, and may refer toa portion of a chromosome, a gene, functional genetic element, or asingle nucleotide or basepair. As used herein in reference to CpG sitesthat may be methylated, a locus refers to the C residue in the CpGdinucleotide.

As used herein, a “collection liquid” is a liquid in which to place asample to preserve, stabilize, and otherwise maintain its integrity as arepresentative sample of the specimen from which the sample was taken.While not limited in the types of compositions that find use ascollection liquids, examples of collection liquids are aqueous buffersoptionally comprising a preservative and organic solvents, such asacetonitrile.

As used herein, “a capture reagent” refers to any agent that is capableof binding to an analyte (e.g., a target). Preferably, “a capturereagent” refers to any agent that is capable of specifically binding toan analyte, e.g., having a higher binding affinity and/or specificity tothe analyte than to any other moiety. Any moiety, such as a cell, acellular organelle, an inorganic molecule, an organic molecule and amixture or complex thereof can be used as a capture reagent if it hasthe requisite binding affinity and/or specificity to the analyte. Thecapture reagents can be peptides, proteins, e.g., antibodies orreceptors, oligonucleotides, nucleic acids, vitamins, oligosaccharides,carbohydrates, lipids, small molecules, or a complex thereof. Capturereagents that comprise nucleic acids, e.g., oligonucleotides, maycapture a nucleic acid target by sequence-specific hybridization (e.g.,through the formation of conventional Watson-Crick basepairs), orthrough other binding interactions. When a capture oligonucleotidehybridizes to a target nucleic acid, hybridization may involve a portionof the oligonucleotide, or the complete oligonucleotide sequence, andthe oligonucleotide may bind to a portion of or to the complete targetnucleic acid sequence.

As used herein, “PVP” refers to polyvinylpyrrolidone, which is awater-soluble polymer made from the monomer N-vinylpyrrolidone. The termPVP is used herein to refer to PVP in various states of cross-linkedpolymerization, including preparations of PVP that may also be known inthe art as polyvinylpolypyrrolidone (PVPP).

As used herein, a “magnet” is a material or object that produces amagnetic field. A magnet may be a permanent magnet or an electromagnet.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S.Pat. No. 5,494,810; herein incorporated by reference in its entirety)are forms of amplification. Additional types of amplification include,but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No.5,639,611; herein incorporated by reference in its entirety), assemblyPCR (see, e.g., U.S. Pat. No. 5,965,408; herein incorporated byreference in its entirety), helicase-dependent amplification (see, e.g.,U.S. Pat. No. 7,662,594; herein incorporated by reference in itsentirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and5,338,671; each herein incorporated by reference in their entireties),intersequence-specfic PCR, inverse PCR (see, e.g., Triglia, et al et al.(1988) Nucleic Acids Res., 16:8186; herein incorporated by reference inits entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al etal., Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No.5,508,169; each of which are herein incorporated by reference in theirentireties), methylation-specific PCR (see, e.g., Herman, et al., (1996)PNAS 93(13) 9821-9826; herein incorporated by reference in itsentirety), miniprimer PCR, multiplex ligation-dependent probeamplification (see, e.g., Schouten, et al., (2002) Nucleic AcidsResearch 30(12): e57; herein incorporated by reference in its entirety),multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic AcidsResearch 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80; each of whichare herein incorporated by reference in their entireties), nested PCR,overlap-extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic AcidsResearch 16(15) 7351-7367; herein incorporated by reference in itsentirety), real time PCR (see, e.g., Higuchi, et al., (1992)Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology11:1026-1030; each of which are herein incorporated by reference intheir entireties), reverse transcription PCR (see, e.g., Bustin, S. A.(2000) J. Molecular Endocrinology 25:169-193; herein incorporated byreference in its entirety), solid phase PCR, thermal asymmetricinterlaced PCR, and Touchdown PCR (see, e.g., Don, et al., Nucleic AcidsResearch (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5)812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; each ofwhich are herein incorporated by reference in their entireties).Polynucleotide amplification also can be accomplished using digital PCR(see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004,(1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41,(1999); International Patent Publication No. WO05023091A2; US PatentApplication Publication No. 20070202525; each of which are incorporatedherein by reference in their entireties).

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic or other DNA or RNA, withoutcloning or purification. This process for amplifying the target sequenceconsists of introducing a large excess of two oligonucleotide primers tothe DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (“PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified” and are “PCR products” or “amplicons.” Those of skillin the art will understand the term “PCR” encompasses many variants ofthe originally described method using, e.g., real time PCR, nested PCR,reverse transcription PCR (RT-PCR), single primer and arbitrarily primedPCR, etc.

As used herein, the term “nucleic acid detection assay” refers to anymethod of determining the nucleotide composition of a nucleic acid ofinterest. Nucleic acid detection assay include but are not limited to,DNA sequencing methods, probe hybridization methods, structure specificcleavage assays (e.g., the INVADER assay, (Hologic, Inc.) and aredescribed, e.g., in U.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069,6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009/0253142, each of which is herein incorporated by reference in itsentirety for all purposes); enzyme mismatch cleavage methods (e.g.,Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, hereinincorporated by reference in their entireties); polymerase chainreaction (PCR), described above; branched hybridization methods (e.g.,Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802,herein incorporated by reference in their entireties); rolling circlereplication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502,herein incorporated by reference in their entireties); NASBA (e.g., U.S.Pat. No. 5,409,818, herein incorporated by reference in its entirety);molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, hereinincorporated by reference in its entirety); E-sensor technology(Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and6,063,573, herein incorporated by reference in their entireties);cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and5,660,988, herein incorporated by reference in their entireties); DadeBehring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001,6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated byreference in their entireties); ligase chain reaction (e.g., BaranayProc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridizationmethods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by referencein its entirety).

In some embodiments, target nucleic acid is amplified (e.g., by PCR) andamplified nucleic acid is detected simultaneously using an invasivecleavage assay. Assays configured for performing a detection assay(e.g., invasive cleavage assay) in combination with an amplificationassay are described in US Patent Publication US 20090253142 A1 (App.Ser. No. 12/404,240), incorporated herein by reference in its entiretyfor all purposes. Additional amplification plus invasive cleavagedetection configurations, termed the QuARTS method, are described inU.S. patent application Ser. Nos. 12/946,737; 12/946,745; and12/946,752, incorporated herein by reference in their entireties for allpurposes.

The term “invasive cleavage structure” as used herein refers to acleavage structure comprising i) a target nucleic acid, ii) an upstreamnucleic acid (e.g., an INVADER oligonucleotide), and iii) a downstreamnucleic acid (e.g., a probe), where the upstream and downstream nucleicacids anneal to contiguous regions of the target nucleic acid, and wherean overlap forms between the a 3′ portion of the upstream nucleic acidand duplex formed between the downstream nucleic acid and the targetnucleic acid. An overlap occurs where one or more bases from theupstream and downstream nucleic acids occupy the same position withrespect to a target nucleic acid base, whether or not the overlappingbase(s) of the upstream nucleic acid are complementary with the targetnucleic acid, and whether or not those bases are natural bases ornon-natural bases. In some embodiments, the 3′ portion of the upstreamnucleic acid that overlaps with the downstream duplex is a non-basechemical moiety such as an aromatic ring structure, e.g., as disclosed,for example, in U.S. Pat. No. 6,090,543, incorporated herein byreference in its entirety. In some embodiments, one or more of thenucleic acids may be attached to each other, e.g., through a covalentlinkage such as nucleic acid stem-loop, or through a non-nucleic acidchemical linkage (e.g., a multi-carbon chain).

As used herein, the terms “complementary” or “complementarity” used inreference to polynucleotides (i.e., a sequence of nucleotides) refers topolynucleotides related by the base-pairing rules. For example, thesequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally, as in a purified restriction digest, or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced (e.g., in the presence of nucleotides and an inducing agent suchas a biocatalyst (e.g., a DNA polymerase or the like). The primer istypically single stranded for maximum efficiency in amplification, butmay alternatively be partially or completely double stranded. Theportion of the primer that hybridizes to a template nucleic acid issufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method. Primers may comprise labels, tags, capture moieties,etc.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP).

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.To further illustrate, oligonucleotides are typically less than 200residues long (e.g., between 15 and 100), however, as used herein, theterm is also intended to encompass longer polynucleotide chains.Oligonucleotides are often referred to by their length. For example a 24residue oligonucleotide is referred to as a “24-mer”. Typically, thenucleoside monomers are linked by phosphodiester bonds or analogsthereof, including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions arepresent. Further, oligonucleotides are typically single-stranded.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J AmChem Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, entitled “PROCESS FORPREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al., orother methods known to those skilled in the art. All of these referencesare incorporated by reference.

A “sequence” of a biopolymer refers to the order and identity of monomerunits (e.g., nucleotides, amino acids, etc.) in the biopolymer. Thesequence (e.g., base sequence) of a nucleic acid is typically read inthe 5′ to 3′ direction.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified,” “mutant,” and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA)sequence that comprises coding sequences necessary for the production ofa polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment polypeptide areretained. The term also encompasses the coding region of a structuralgene and the sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb or more on either end suchthat the gene corresponds to the length of the full-length mRNA.Sequences located 5′ of the coding region and present on the mRNA arereferred to as 5′ non-translated sequences. Sequences located 3′ ordownstream of the coding region and present on the mRNA are referred toas 3′ non-translated sequences. The term “gene” encompasses both cDNAand genomic forms of a gene. A genomic form or clone of a gene containsthe coding region interrupted with non-coding sequences termed “introns”or “intervening regions” or “intervening sequences.” Introns aresegments of a gene that are transcribed into nuclear RNA (e g., hnRNA);introns may contain regulatory elements (e.g., enhancers). Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of nucleic acid purificationsystems and reaction assays, such delivery systems include systems thatallow for the storage, transport, or delivery of reagents and devices(e.g., inhibitor adsorbants, particles, denaturants, oligonucleotides,spin filters etc. in the appropriate containers) and/or supportingmaterials (e.g., buffers, written instructions for performing aprocedure, etc.) from one location to another. For example, kits includeone or more enclosures (e.g., boxes) containing the relevant reactionreagents and/or supporting materials. As used herein, the term“fragmented kit” refers to a delivery system comprising two or moreseparate containers that each contains a subportion of the total kitcomponents. The containers may be delivered to the intended recipienttogether or separately. For example, a first container may contain anmaterials for sample collection and a buffer, while a second containercontains capture oligonucleotides and denaturant. The term “fragmentedkit” is intended to encompass kits containing Analyte specific reagents(ASR's) regulated under section 520(e) of the Federal Food, Drug, andCosmetic Act, but are not limited thereto. Indeed, any delivery systemcomprising two or more separate containers that each contains asubportion of the total kit components are included in the term“fragmented kit.” In contrast, a “combined kit” refers to a deliverysystem containing all of the components of a reaction assay in a singlecontainer (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

The term “system” as used herein refers to a collection of articles foruse for a particular purpose. In some embodiments, the articles compriseinstructions for use, as information supplied on e.g., an article, onpaper, or on recordable media (e.g., diskette, CD, flash drive, etc.).In some embodiments, instructions direct a user to an online location,e.g., a website.

As used herein, the term “information” refers to any collection of factsor data. In reference to information stored or processed using acomputer system(s), including but not limited to internets, the termrefers to any data stored in any format (e.g., analog, digital, optical,etc.). As used herein, the term “information related to a subject”refers to facts or data pertaining to a subject (e.g., a human, plant,or animal). The term “genomic information” refers to informationpertaining to a genome including, but not limited to, nucleic acidsequences, genes, percentage methylation, allele frequencies, RNAexpression levels, protein expression, phenotypes correlating togenotypes, etc. “Allele frequency information” refers to facts or datapertaining to allele frequencies, including, but not limited to, alleleidentities, statistical correlations between the presence of an alleleand a characteristic of a subject (e.g., a human subject), the presenceor absence of an allele in an individual or population, the percentagelikelihood of an allele being present in an individual having one ormore particular characteristics, etc.

EMBODIMENTS OF THE TECHNOLOGY

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

1. Methods Generally

Provided herein are methods for isolating DNA, for example, from a stoolsample. As summarized in FIG. 1, the process comprises homogenizing asample (e.g., a stool sample) in a suitable buffer and preparing asupernatant from the homogenate. The supernatant is treated with acomposition (e.g., a cross-linked polyvinylpyrrolidone (PVP) such aspolyvinylpolypyrrolidone (PVPP)) to remove inhibitors and produce aclarified supernatant. DNA in the clarified supernatant is denatured,e.g., by adding guanidine thiocyanate (GTC) and/or by heating thesample. Then, a target capture reagent, e.g., a magnetic bead to whichis linked an oligonucleotide complementary to the target, is added andthe solution is incubated under conditions (e.g., ambient temperaturefor an hour) that promote the association (e.g., by hybridization) ofthe target with the capture reagent to produce a target:capture reagentcomplex. After isolating and removing the target:capture reagent complex(e.g., by application of a magnetic field), the resulting solution isheated again to denature the remaining DNA in the clarified supernatantand another target capture reagent can be added to isolate anothertarget. The process can be repeated, e.g., at least four times, toisolate as many targets as are required for the assay (e.g., asequential or serial extraction). The isolated target:capture reagentcomplexes from each capture and isolation step are washed and the targetDNAs are eluted using a small volume of buffer suitable for downstreamanalysis.

2. Inhibitor Removal

The sample may be a sample of material that contains impurities thatbreak down nucleic acids or inhibit enzymatic reactions. In particular,such impurities inhibit the catalytic activity of enzymes that interactwith nucleic acids, e.g., nucleases such as restriction endonucleases,reverse transcriptases, nucleic acid polymerases, ligases, etc.,particularly enzymes that are used for polymerase chain reaction (PCR),LCR (ligase chain reaction), TMA (transcription-mediated amplification),NASBA (nucleic acid base specific amplification), 3SR (self-sustainedsequence replication), and the like.

2.1 PVP

In some embodiments, inhibitors in a sample are removed by treatmentwith polyvinylpyrrolidone (see also, e.g., U.S. Pat. Appl. Ser. No.61/485,338, which is incorporated herein by reference).Polyvinylpyrrolidone (PVP) is a water-soluble polymer made from themonomer N-vinylpyrrolidone (see FIG. 2). Polyvinylpolypyrrolidone (PVPP)is a highly cross-linked modification of PVP. The extent ofcross-linking varies and there is no defined threshold establishing adivision between PVP and PVPP. Accordingly, the term PVP is used hereinto refer to PVP in various states of cross-linked polymerization,including preparations of PVP that may also be known in the art as PVPP.An important property, however, is that as the extent of cross-linkingis increased, the polymer becomes increasingly insoluble in water. Thecross-linked forms absorb water, which causes the polymer to swell. Thesynthesis and physical properties of PVP and PVPP are well-known in theart (e.g., see Haaf, Sanner, & Straub. Polymers of N-vinylpyrrolidone:synthesis, characterization, and uses. Polymer J. 17(1): 143 (1985)).

PVP has been used in many technical applications including use as ablood plasma expander; as a binder in many pharmaceutical tablets; as anadhesive in glue stick and hot melts; as an additive for batteries,ceramics, fiberglass, inks, inkjet paper and in the chemical-mechanicalplanarization process; as an emulsifier and disintegrant for solutionpolymerization; as photoresist; for production of membranes, such asdialysis and water purification filters; as a thickening agent in toothwhitening gels, etc.

PVP has also found use in binding impurities and removing them fromsolutions, particularly in wine-making and beer-making to removepolyphenols (see, e.g., Redmanji, Gopal, & Mola. A novel stabilizationof beer with Polyclar Brewbrite. MBAA TQ 39(1): 24 (2002)). The use ofsoluble and insoluble forms of PVP has been described in relation toprocessing biological samples, for example, as a means to neutralizephenols (see, e.g., U.S. Pat. No. 7,005,266; Shames, et al.Identification of widespread Helicobacter hepaticus infection in fecesin commercial mouse colonies by culture and PCR assay. J. Clin.Microbiol. 33(11): 2968 (1995); Morgan et al. Comparison of PCR andmicroscopy for detection of Cryptosporidium parvum in human fecalspecimens: Clinical trial. J. Clin. Microbiol. 36(4): 995 (1998)).

The PVP is provided in forms that allow its introduction into a samplethat is to be processed, e.g., as a powder, slurry, suspension, ingranules, and the like. In some embodiments of the technology providedherein, the PVP is provided premeasured in a ready-to-use form. Forexample, in some embodiments, the PVP is pressed into a tabletcomprising the mass of PVP appropriate for treating a sample. Differentsizes and shapes of tablets are provided for different volumes and typesof samples. Inert binders, fillers, and other compositions may be addedto the tablets to provide physical, thermal, chemical, and biologicalstability, or to provide other desired characteristics such as improveddispersion within the sample or controlled-release.

Both the degree of cross-linking and the size of the PVP particles areparameters affecting the downstream assay of the resulting nucleic acidpreparations. For example, soluble PVP has been found to inhibit somedownstream assays. Accordingly, the method benefits from using a PVPthat is sufficiently insoluble (e.g., sufficiently cross-linked) toallow adequate removal of the PVP by downstream processing steps (e.g.,centrifugation and/or spin filtration). In addition, when thecross-linked PVP particles are too small they pack too tightly in thespin column and restrict the effluent flow of the sample into the spincolumn collection space. For example, experiments performed during thedevelopment of some embodiments of the present technology demonstratedthat a PVP having an average particle size of 100-130 micrometersproduced satisfactory results while a PVP having an average particlesize of 30-50 micrometers restricted flow and filtration. Furtherexperimentation may indicate that other sizes and solubilities may beappropriate for embodiments of the method.

2.2 Spin Filter

The technology provided herein encompasses use of a spin filter, forexample, as provided in U.S. Pat. Appl. Ser. No. 61/485,214 to filterPVP-treated samples treated to remove inhibitors bound to the PVP. Asdiscussed above, during the development of the PVP treatment method,experiments demonstrated that conventional spin columns having a filterfrit in the bottom end clogged under some conditions. Accordingly, someembodiments of the technology comprise using a clog-resistant spinfilter. FIGS. 3-8 depict various configurations of a clog-resistant spinfilter in assembled and exploded views and associated with a collectiontube. The clog-resistant filter is designed to allow the sample to befiltered through the body walls if the bottom end becomes clogged withresidue from the sample.

Spin filters appropriate for use with the technology provided herein aregenerally made from a material is inert with respect to the sample—thatis, the material does not react with or otherwise contaminate or modifythe sample, other than filtering it, in a way that affects a subsequentassay (e.g., causes degradation of the sample, causes its decomposition,or the like). An example of such a material is polyethylene. Othersuitable materials are, e.g., nylon, cellulose-acetate,polytetrafluoroethylene (PTFE, also known as Teflon), polyvinylidenefluoride (PVDF), polyester, and polyethersulfone. Operating pressure,the chemical and physical characteristics of the composition to befiltered, the size of the entity to remove from the sample, and themechanical properties of the material (e.g., capability to withstandcentrifugation at the speed required for the filtering application) arefactors that are considered when selecting an appropriate spin filter.

Filters are manufactured to have various pore sizes appropriate fordifferent filtering applications. For example, a filter with pore sizeof 0.2 micrometers is typically acknowledged to remove most bacteriawhile smaller pore sizes are required to remove viruses and bacterialspores. For removing larger particulates, a larger pore size isadequate. For example, while one aspect of the technology providedherein uses a spin filter having a 20-micrometer pore size, other poresizes that find use in filtration applications are 0.22, 0.45, 10, 20,30, and 45 micrometers. Accordingly, larger and smaller pore sizes arecontemplated, as well as pore sizes intermediate within the intervalsdelimited by these particular values. For some filtration applicationsthe filter is characterized by the average molecular weight of themolecules that are retained by the filter. For example, a filter with a5,000 Da molecular weight cutoff (MWCO) is designed to retain moleculesand complexes having at least a molecular weight of approximately 5,000Da. Filters can provide MWCOs of 10,000 Da; 30,000 Da; 50,000 Da;100,000 Da, and other limits required for the filtration task. Operatingpressure and the size of the entity to remove from the sample arefactors to consider when choosing a pore size or cutoff value.

3. Nucleic Acid Capture

The target nucleic acids are captured using a sequence-specific targetcapture reagent, e.g., a magnetic bead to which is linked anoligonucleotide complementary to the target. After adding the capturereagent, the solution is incubated under conditions that promote theassociation (e.g., by hybridization) of the target with the capturereagent to produce a target:capture reagent complex. After isolating andremoving the target:capture reagent complex (e.g., by application of amagnetic field), the resulting solution is heated again to denature theremaining DNA in the clarified supernatant and another target capturereagent can be added to isolate another target (e.g., by hybridizationand application of a magnetic field). The process can be repeated, e.g.,at least four times, to isolate as many targets as are required for theassay (e.g., a sequential or serial isolation process as described,e.g., by U.S. Pat. Appl. Ser. No. 61/485,386, which is incorporatedherein by reference). Also, more than one target can be isolated in acapture step by using a capture reagent comprising multiple capturesequences.

3.1 Capture Reagents

In one aspect, the methods provided herein relate to the use of capturereagents. Such reagents are molecules, moieties, substances, orcompositions that preferentially (e.g., specifically and selectively)interact with a particular target sought to be isolated and purified.Any capture reagent having desired binding affinity and/or specificityto the analyte target is used in the present technology. For example, insome embodiments the capture reagent is a macromolecule such as apeptide, a protein (e.g., an antibody or receptor), an oligonucleotide,a nucleic acid, (e.g., nucleic acids capable of hybridizing with thetarget nucleic acids), a vitamin, an oligosaccharide, a carbohydrate, alipid, or a small molecule, or a complex thereof. As illustrative andnon-limiting examples, an avidin target capture reagent may be used toisolate and purify targets comprising a biotin moiety, an antibody maybe used to isolate and purify targets comprising the appropriate antigenor epitope, and an oligonucleotide may be used to isolate and purify acomplementary oligonucleotide (e.g., a poly-dT oligonucleotide may beused to isolate and purify targets comprising a poly-A tail).

Any nucleic acids, including single-, double-, and triple-strandednucleic acids, that are capable of binding, or specifically binding, tothe target are used as the capture reagent in the present device.Examples of such nucleic acids include DNA, such as A-, B- or Z-formDNA, and RNA such as mRNA, tRNA and rRNA, aptamers, peptide nucleicacids, and other modifications to the sugar, phosphate, or nucleosidebase. Thus, there are many strategies for capturing a target andaccordingly many types of capture reagents are known to those in theart. While not limited in the means by which a target nucleic acid canbe captured, embodiments of the technology provided herein compriseusing an oligonucleotide that is complementary to the target and thatthus captures the target by specifically and selectively hybridizing tothe target nucleic acid.

In addition, target capture reagents comprise a functionality tolocalize, concentrate, aggregate, etc. the capture reagent and thusprovide a way to isolate and purify the target when captured (e.g.,bound, hybridized, etc.) to the capture reagent, e.g., when atarget:capture reagent complex is formed. For example, in someembodiments the portion of the target capture reagent that interactswith the target (e.g., the oligonucleotide) is linked to a solid support(e.g., a bead, surface, resin, column) that allows manipulation by theuser on a macroscopic scale. Often, the solid support allows the use ofa mechanical means to isolate and purify the target:capture reagentcomplex from a heterogeneous solution. For example, when linked to abead, separation is achieved by removing the bead from the heterogeneoussolution, e.g., by physical movement. In embodiments in which the beadis magnetic or paramagnetic, a magnetic field is used to achievephysical separation of the capture reagent (and thus the target) fromthe heterogeneous solution. Magnetic beads used to isolate targets aredescribed in the art, e.g., as described in U.S. Pat. No. 5,648,124 andEuropean Pat. Appl. No. 87309308, incorporated herein by reference intheir entireties for all purposes.

In some embodiments, the component of the capture reagent that interactswith the target (e.g., an oligonucleotide) is attached covalently to thecomponent of the capture reagent that provides for the localization,concentration, and/or aggregation (e.g., the magnetic bead) of thetarget:capture reagent complex. Exemplary embodiments of suchcovalently-linked capture reagents are provided by Stone, et al.(“Detection of rRNA from four respiratory pathogens using an automatedQβ replicase assay”, Molecular and Cellular Probes 10: 359-370 (1996)),which is incorporated herein by reference in its entirety for allpurposes. These covalently-linked capture reagents find use in thesequential isolation of multiple specific targets from the same samplepreparation. Moreover, these capture reagents provide for the isolationof DNA targets without many of the problems that are associated withother methods. For example, the use of a conventional streptavidin beadto capture a biotinylated target is problematic for processing samplesthat comprise large amounts of free biotin (e.g., a stool sample)because the free biotin interferes with isolation of the target.

3.2 Magnetic Particle Localizer

The target:capture reagent complexes are captured using a magneticparticle localizer. However, sample viscosity can have a profound effecton localization efficiency due to the viscous drag affecting themagnetic microparticles. Stool samples have viscosities ranging from 20centipoise to 40 centipoise, whereas, for reference, water at 20° C. hasa viscosity of approximately 1 centipoise and honey at 20° C. has aviscosity of approximately 3,000 centipoise. Thus, for someapplications, stronger magnetic fields may be preferred in order toprovide for a more efficient isolation.

It has been found that particularly efficient isolations are obtainedusing magnetic devices having particular arrangements of magnets. Forexample, one particularly effective arrangement provides two sets ofmagnets circularly arranged in parallel planar layers around the sample,with the magnets in one layer oriented all with their north poles towardthe sample and the magnets in the other layer are all oriented withtheir south poles toward the sample (i.e., the “N-S” configuration, asopposed to other orientations such as the “N-N” orientation in which allnorth poles or all south poles in both layers are oriented toward thesample). An example of such a device is provided by Light and Miller,U.S. patent application Ser. No. 13/089,116 (“Magnetic MicroparticleLocalization Device”), which is incorporated herein in its entirety forall purposes. In some configurations, the magnets of the device arearranged around a hole into which a sample tube (e.g., a 50 milliliterconical tube) is placed, such that they produce a magnetic flux in thesample. The magnetic flux effects the movement of the magnetic particlesin the solution such that they are aggregated, concentrated, and/orisolated in an area of the sample tube that facilitates removal of therecovery of the target DNA (Light, supra).

Such devices have shown to be particularly effective for thelocalization of magnetic particles in large, viscous samples (e.g.,stool samples) and thus are useful for the isolation of DNA from suchsamples (Light, supra). For example, FIGS. 11A and 11B show the effectof sample viscosity on the clearance of magnetic beads from solutions of1 or 25 centipoise viscosity using conventional magnetic technology(11A) or the magnetic localization technology of Light and Miller (11B)(Light, supra). In the graphs shown, a decrease in absorbance indicatesa decreased concentration of microparticles suspended in solution. Thedata collected for the 25 centipoise solutions are shown with squares(▪) and data collected for the 1 centipoise solution are shown withdiamonds (♦). These graphs show that the increase in viscosity slows theseparation dramatically when conventional technology is used, while theLight and Miller magnetic particle localization device clears the moreviscous solution with only a modest reduction in speed.

The chemistries and processes described above, when used in combination,provide a system for the isolation of nucleic acids from complex andinhibitory samples, such as stool samples, that is significantly fasterthan previously used methods. Moreover, the system produces nucleic acidpreparations that are substantially more free of inhibitory substancesand results in a higher yield of target nucleic acid for, e.g.,diagnostic testing. Further, embodiments of this system are readilyintegrated into the laboratory workflow for efficient sample processingfor use with any downstream analysis or detection technology. Acomparison of the workflow, timeline, and process yields of anembodiment of the instant system and an exemplary conventional system isshown in FIG. 14.

4. Kits

It is contemplated that embodiments of the technology are provided inthe form of a kit. The kits comprise embodiments of the compositions,devices, apparatuses, etc. described herein, and instructions for use ofthe kit. Such instructions describe appropriate methods for preparing ananalyte from a sample, e.g., for collecting a sample and preparing anucleic acid from the sample. Individual components of the kit arepackaged in appropriate containers and packaging (e.g., vials, boxes,blister packs, ampules, jars, bottles, tubes, and the like) and thecomponents are packaged together in an appropriate container (e.g., abox or boxes) for convenient storage, shipping, and/or use by the userof the kit. It is understood that liquid components (e.g., a buffer) maybe provided in a lyophilized form to be reconstituted by the user. Kitsmay include a control or reference for assessing, validating, and/orassuring the performance of the kit. For example, a kit for assaying theamount of a nucleic acid present in a sample may include a controlcomprising a known concentration of the same or another nucleic acid forcomparison and, in some embodiments, a detection reagent (e.g., aprimer) specific for the control nucleic acid. The kits are appropriatefor use in a clinical setting and, in some embodiments, for use in auser's home. The components of a kit, in some embodiments, provide thefunctionalities of a system for preparing a nucleic acid solution from asample. In some embodiments, certain components of the system areprovided by the user.

EXAMPLES Example 1

During the development of embodiments of the technology provided herein,it was demonstrated that PVP (e.g., PVPP) removes PCR inhibitors from astool sample (see FIG. 9). Volumes of 20 milliliters were taken from thesupernatants of two different stool supernatant samples. For each stoolsample, one aliquot was treated with PVP and the other was leftuntreated. Otherwise, the samples were processed identically to capturetwo different nucleic acid targets (FIG. 9, Gene A and Gene V). Aftercapture and final elution, the recoveries of the two targets weremonitored by a SYBR Green quantitative PCR (qPCR) assay using 1microliter of eluate in a 25 microliter reaction volume. For bothtargets from both stool supernatants, aliquots treated with PVP wereamplified whereas the untreated aliquots failed to produce any qPCRsignal. These results demonstrate the necessity and efficacy of PVP asan inhibitor-removal treatment when extracting DNA from stool samplesfor assay by a quantitative PCR assay.

Example 2

During the development of embodiments of the technology provided herein,data were collected demonstrating that spin filtering improves theremoval of PCR inhibitors. The experiment compared PVP (e.g., PVPP) ofdifferent sizes for the ability to remove PCR inhibitors from stoolsupernatant samples. Two commercially available PVP compositions werecompared: Polyclar® 10 and Polyplasdone® XL, which are composed of PVPparticles having an average diameter of 30-50 micrometers and 100-130micrometers, respectively. Inhibitor removal by the two PVP compositionswas assessed by qPCR in which 1 microliter or 5 microliters of theisolated DNA eluates were used in a 25-microliter reaction volume.First, both types of PVP were separated from the stool supernatant bypelleting (centrifugation). For both PVP types, samples showed equalrecovery and amplification curve shape when 1 microliter of eluted DNAwas added to the qPCR. However, using 5 microliters of eluate failed toproduce any qPCR signal, indicating that PCR inhibitors remained in thesample (see FIGS. 10A and 10B).

Next, spin column filtration was tried as an alternative method toseparate the PVP from the stool supernatant. The smaller particle sizePVP could not be processed in this manner as the PVP apparently packeddown so tightly in the spin column that the liquid stool supernatantcould not pass through. However, the larger particle size PVP did nothave this same problem and the sample preparation could easily be spinfiltered. The spin column contained a polyethylene frit (20-micrometernominal pore size) to collect the PVP. When separating the largeparticle PVP from the stool supernatant via spin column filtrationequipped with a polyethylene frit, the eluate volume in the qPCR couldbe increased to 5 microliters or 6 microliters without obviousinhibition (see FIGS. 10C and 10D). As shown in Table 1, when using 5 or6 microliters of eluate, the calculated strand number was approximatelyfive or six times the calculated strand number when using 1 microliterof eluate. These results demonstrate the benefits of PVP treatment plusspin column filtration for removal of PCR inhibitors from stool samples.

TABLE 1 Treatment Volume Strands % Expected PVPP 30-50 1 μL  950 No spinfilter 5 μL No Signal 0 (complete inhibition) PVPP 100-130 1 μL  907 Nospin filter 5 μL No Signal 0 (complete inhibition) PVPP 100-130 1 μL1136 With spin filter 5 μL 6751 119 PVPP 100-130 1 μL 3110 With spinfilter 6 μL 18600  99.68

Example 3

During the development of embodiments of the technology provided herein,experiments were performed to compare the localization efficiencies ofthe conventional technology (e.g., a Promega PolyA Tract backed with a1-inch outer diameter×one-eighth-inch thick N52 neodymium magnet) andthe magnetic microparticle localizing device of Light and Miller (gradeN52 neodymium magnets in the S-N configuration) for samples of low(i.e., 1 centipoise) and high (i.e., 25 centipoise) viscosities.

Test solutions of the appropriate viscosity (e.g., 1 or 25 centipoise)were placed in a conventional device or an embodiment of the technologyprovided herein for testing. Samples were exposed to the magnetic field,the liquid was aspirated at the time intervals indicated for eachsample, and the particles remaining in suspension were quantified byspectrometry. A decrease in absorbance indicates a decreasedconcentration of microparticles suspended in solution (i.e., moreparticles localized and removed from suspension by magnetic separation).Results for the conventional technology are provided below in FIG. 11A.Results for the magnetic microparticle localization device are providedin FIG. 11B. In FIGS. 11A and B, data collected for the 25 centipoisesolution are shown with squares (▪) and data collected for the 1centipoise solution are shown with diamonds (♦).

Example 4

During the development of embodiments of the technology provided herein,it was demonstrated that the majority of the DNA for a given target isdepleted from a stool supernatant in a single extraction. The extractionwas performed according to the flow chart shown in FIG. 1. After finalelution, the recoveries of the two targets (Gene A and Gene V) fromextractions 1 and 2 were monitored by SYBR Green qPCR assays using 1microliter of eluate in a 25-microliter volume reaction. For bothtargets, extraction 1 yielded good recovery of target, whereas theeluate from extraction 2 failed to produce any qPCR signal for eithertarget (FIG. 12).

Example 5

During the development of embodiments of the technology provided herein,it was demonstrated that DNA extraction can be performed repeatedly on asingle sample through a minimum of four cycles ofdenaturation/hybridization without compromising the integrity of thehuman DNA in the stool supernatant. In this example, four targets (GenesA, F, V, and W) were captured from the sample and the order of theircapture was varied. After elution, the recovery of each target wasmonitored by SYBR Green qPCR. In FIG. 13, plots show the amplificationcurves for each gene when it was captured first, second, third, andfourth in the extraction sequence. The superposition of theamplification curves demonstrates that recoveries were approximatelyequal regardless of the order of extraction. Table 3 quantifies theresults from FIG. 13.

TABLE 3 Target Extraction Mean C_(p) Mean Strands/μL Gene A #1 28.92 862#2 28.89 878 #3 28.85 907 #4 28.73 984 Gene F #1 29.32 499 #2 29.36 489#3 29.29 511 #4 29.01 614 Gene V #1 31.29 129 #2 31.01 155 #3 31.18 139#4 30.84 177 Gene W #1 29.17 724 #2 29.11 757 #3 28.99 819 #4 29.16 730

For all four genes, the mean C_(p) (Crossing point—the cycle number atwhich the amplification curve crosses a fixed threshold) and strandnumbers were essentially equal regardless of the order of extraction.

Example 6

Exemplary Procedure for Serial Isolation of a Plurality of TargetNucleic acids:

As diagrammed in FIG. 1:

-   -   1. A stool sample is homogenized, e.g., with a buffer, to form a        stool homogenate. The homogenate treated to partition residual        solids from the fluid, e.g., by centrifugation or filtration, to        produce a “stool supernatant.”    -   2. Stool supernatant is treated to remove assay inhibitors        (e.g., with polyvinylpolypyrrolidone, as described in U.S. Pat.        Appl. Ser. No. 61/485,338, which is incorporated herein by        reference in its entirety), producing “clarified supernatant”.    -   3. Ten milliliters of clarified supernatant (representing an        equivalent of approximately 4 grams of stool) is mixed with        guanidine thiocyanate (GTC) to a final concentration of 2.4 M;    -   4. The mixture is then heated in a 90° C. water bath for 10        minutes to denature the

DNA (and proteins) present in the stool.

-   -   5. Paramagnetic particles containing covalently attached        (coupled) oligonucleotides complementary to the target        sequence(s) of interest (“target-specific capture probes”) are        added to the sample. The sample is then incubated (e.g., at        ambient temperature, about 22-25° C.) for one hour to enable        hybridization of the target DNA to the capture probes on the        magnetic particles.    -   6. The mixture of clarified supernatant, GTC, and particles is        exposed to a magnetic field to separate the particles (now        containing target DNA hybridized to the capture probes) from the        stool supernatant/GTC mixture, which is transferred to a new        tube. See, e.g., U.S. patent application Ser. No. 13/089,116,        which is incorporated herein by reference.    -   7. The paramagnetic particles are then washed and the target DNA        eluted, ready for use in detection assays.    -   8. The supernatant/GTC mixture retained in step 6 is returned to        the 90° C. water bath for 10 minutes to repeat denaturation        (step 4). Step 5 is then repeated by adding magnetic particles        containing capture probes complementary to different targets        DNAs, and the hybridization, particle separation and elution        steps are repeated to produce a purified sample of a second DNA        target.

The denaturation/hybridization/separation cycle (steps 4-6) can berepeated at least four or more times to serially extract differenttarget DNAs from the same stool supernatant sample.

Example 7

During the development of embodiments of the technology provided herein,the methods were tested in a clinical application. The followingprovides an example of workflow using the systems and methods of thepresent invention.

Study Design

This study was based on well-characterized archival stools from multiplemedical centers, including referral centers and community medicalcenters in the United States and Denmark. Approval by institutionalreview boards was obtained. Stools were procured from case patients withproven colorectal cancer (CRC), cases with at least one colorectaladenoma≧1 centimeter, and age and sex matched control patients withoutneoplasia as assessed by colonoscopy. Patients had been recruited fromboth clinical and screening settings, and some were symptomatic. Thosewith known cancer syndromes or inflammatory bowel disease were excluded.Nearly 700 samples were tested, of which 133 were adenomas≧1 centimeterand 252 were cancer patients.

A multi-marker stool test was performed that included four methylatedgenes (vimentin, NDRG4, BMP3, and TFPI2), mutant KRAS, a reference genebeta-actin (ACTB), and hemoglobin. To evaluate test performance, caseand control stools were distributed in balanced fashion to two differenttest sites; all assays were run by blinded technicians.

Stool Collection and Storage.

Prior to colonoscopy, which served as the gold standard, whole stoolswere collected in plastic buckets. A preservative buffer was added tothe stool and buffered stools were archived at −80° C. However, thetiming of buffer addition, duration between defecation and freezing, andwhether or not samples were homogenized prior to storage were notstandardized and varied across participating centers.

Marker Selection.

Candidate genes were identified that individually or in combinations(e.g., KRAS+BMP3+NDRG4+TFPI2+vimentin+reference and/or ACTB+hemoglobin)yielded nearly complete separation of colorectal neoplasia from normalmucosa. Four methylated gene markers emerged as the mostdiscriminant—NDRG4, BMP3, vimentin, and TFPI2. Mutant KRAS andhemoglobin detection complement methylated gene markers detected instool and, accordingly, were also evaluated in the marker panel.Finally, assay of the reference gene beta-actin (ACTB) was used todetermine total human genome equivalents in stool and, as human DNAlevels in stool increase with colorectal neoplasia, to serve as acandidate marker itself.

Stool Processing and Target Gene Capture

Promptly after thawing, buffered stools were thoroughly homogenized andcentrifuged. A 14-milliliter aliquot of stool supernatant was thentreated with polyvinylpolypyrrolidone at a concentration of 50milligrams per milliliter. Direct capture of target gene sequences byhybridization with oligonucleotide probes was performed on supernatantmaterial. Briefly, 10 milliliters of insoluble PVP-treated supernatantwas denatured in 2.4 M guanidine isothiocyanate (Sigma, St. Louis Mo.)at 90° C. for 10 minutes; 300-500 micrograms of Sera-Mag carboxylatemodified beads (ThermoFisher Scientific, Waltham Mass.) functionalizedwith each oligonucleotide capture probe were subsequently added todenatured stool supernatant and incubated at room temperature for onehour. Sera-Mag beads were collected on a magnetic rack and washed threetimes using MOPS washing buffer (10 mM MOPS; 150 mM NaCl, pH 7.5), andthen eluted in 60 microliters of nuclease free water with 20 nanogramsper microliter tRNA (Sigma). In this study, four selected methylatedmarkers, vimentin, NDRG4, BMP3, and TFPI2, and one reference gene ACTB,were captured together in one hybridization reaction; the mutationmarker KRAS was subsequently captured in another hybridization reaction.The capture probes used, shown here with their 5′-six carbon aminomodified linkage (Integrated DNA Technology, Coralville, Iowa), were asfollows:

for vimentin: (SEQ ID NO: 1)/5AmMC6/CTGTAGGTGCGGGTGGACGTAGTCACGTAGCTCCGGCTGGA-3′; for NDRG4:(SEQ ID NO: 2)/5AmMC6/TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG-3′; for BMP3:(SEQ ID NO: 3) /5AmMC6/GCGGGACACTCCGAAGGCGCAAGGAG-3′; for TFPI2:(SEQ ID NO: 4) /5AmMC6/CGCCTGGAGCAGAAAGCCGCGCACCT-3′; for ACTB:(SEQ ID NO: 5) /5AmMC6/CCTTGTCACACGAGCCAGTGTTAGTACCTACACC-3′; for KRAS:(SEQ ID NO: 6) /5AmMC6/GGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGC-3′and (SEQ ID NO: 7)/5AmMC6/CTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATTAGC-3′

Methylation Assays.

Methylated markers were quantified by the QuARTS method, as we havepreviously described (see, e.g., U.S. patent application Ser. Nos.12/946,737; 12/946,745; and 12/946,752, incorporated herein by referencein their entireties for all purposes). This method combines apolymerase-based target DNA amplification process with an invasivecleavage-based signal amplification process. We treated 45 microlitersof captured DNA with bisulfite using the EZ-96 DNA Methylation Kit (ZymoResearch, Irvine Calif.) and eluted the sample in 50 microliters of 10mM Tris, 0.1 mM EDTA pH 8.0 with 20 nanograms per microliter tRNA(Sigma) on a 96-well PCR plate; 10 microliters of bisulfite-treated DNAwas assayed with the QuARTS method in 30-microliter reaction volumes ona 96-well PCR plate. PCR plates were cycled in a LightCycler 480(Roche).

Two separate triplex QuARTS assays were designed to detect themethylated markers vimentin, NDRG4, BMP3, and TFPI2 using ACTB as areference gene for each. The first triplex assay contained ACTB,vimentin, and NDRG4, and the second contained ACTB, BMP3, and TFPI2.Each QuARTS reaction incorporated 400-600 nM primers and detectionprobes, 100 nM invasive oligonucleotide, 600-700 nM each of FAM(Hologic, Madison Wis.), Yellow (Hologic), and Quasor 670 (BioSearchTechnologies, Novato Calif.) fluorescence resonance energy transferreporter cassettes (FRETs), 6.675 nanogram per microliter Cleavase 2.0(Hologic), 1 unit hot-start GoTaq DNA polymerase (Promega, MadisonWis.), 10 mM MOPS, 7.5 mM MgCl₂, and 250 μM each dNTP. QuARTS cyclingconditions consisted of 95° C. for 3 minutes, then 10 cycles eachcomprising 95° C. for 20 seconds, 67° C. for 30 seconds, and 70° C. for30 seconds, followed by 45 cycles each comprising 95° C. for 20 seconds,53° C. for 1 minute, and 70° C. for 30 seconds, and finally a 30-secondhold at 40° C. For each target below, the two methylation-specificprimers and probe (Integrated DNA Technology, Coralville, Iowa) were asfollows:

For vimentin: Primer (SEQ ID NO: 8) 5′-GGC GGT TCG GGT ATC G-3′, Primer(SEQ ID NO: 9) 5′-CGT AAT CAC GTA ACT CCG AC T-3′, Probe (SEQ ID NO: 10)5′-GAC GCG GAG GCG AGT CGG TCG/3′C6/; for NDRG4: Primer (SEQ ID NO: 11)5′-CGG TTT TCG TTC GTT TTT TCG-3′, Primer (SEQ ID NO: 12)5′-GTA ACT TCC GCC TTC TAC GC-3′, Probe (SEQ ID NO: 13)5′-CGC CGA GGG TTC GTT TAT CG/3′C6/; for BMP3: Primer (SEQ ID NO: 14)5′-GTT TAA TTT TCG GTT TCG TCG TC-3′, Primer (SEQ ID NO: 15)5′-CTC CCG ACG TCG CTA CG-3′, Probe (SEQ ID NO: 16)5′-CGC CGA GGC GGT TTT TTG CG/3′C6/; and for TFPI2: Primer(SEQ ID NO: 17) 5′-TCG TTG GGT AAG GCG TTC-3′, Primer (SEQ ID NO: 18)5′-AAA CGA ACA CCC GAA CCG-3′, Probe (SEQ ID NO: 19)5′-GAC GCG GAG GCG GTT TTT TGT T/3′C6/.

The TFPI2 assay had a specific invasive oligonucleotide:

(SEQ ID NO: 20) 5′-GCG GGA GGA GGT GCC-3′.

Primers and probe for detecting bisulfite-treated ACTB were:

Primer (SEQ ID NO: 21) 5′-TTT GTT TTT TTG ATT AGG TGT TTA AGA-3′, Primer(SEQ ID NO: 22) 5′-CAC CAA CCT CAT AAC CTT ATC-3′, Probe (SEQ ID NO: 23)5′-CCA CGG ACG ATA GTG TTG TGG/ 3′C6/.

Each plate included bisulfite-treated DNA samples, standard curvesamples, positive and negative controls, and water blanks Standardcurves were made using 300 to 1000 target sequences cut from engineeredplasmids. Bisulfite-treated CpGenome universal methylated DNA(Millipore, Billerica, Mass.) and human genomic DNA (Merck, Germany)were used as positive and negative controls. DNA strand number wasdetermined by comparing the C_(p) of the target gene to the standardcurve for the relevant assay. Percent methylation for each marker wasdetermined by dividing the strand number of the methylated gene by theACTB strand number and multiplying by 100.

KRAS Mutation

The KRAS gene was first PCR amplified with primers flanking codons 12/13using 10 microliters of captured KRAS DNA as template. PCR was conductedwith 1× LightCycler® 480 SYBR Green I Master (Roche, Germany) and 200 nMeach primer. Cycling conditions were 95° C. for 3 minutes, followed by15 cycles each at 95° C. for 20 seconds, 62° C. for 30 seconds, and 72°C. for 30 seconds. Primer sequences were:

(SEQ ID NO: 24) 5′-AGG CCT GCT GAA AAT GAC TG-3′, and (SEQ ID NO: 25)5′-CTA TTG TTG GAT CAT ATT CG TC- 3′.

Each amplified sample was diluted 500-fold in nuclease free water. A10-microliter aliquot of the 500-fold sample dilutions was added to a96-well PCR plate with an automated liquid handler (epMotion, Eppendorf,Hauppauge N.Y.). QuARTS assays were then used to evaluate sevenmutations at codons 12/13 of the KRAS gene. Each mutation assay wasdesigned as a singleplex assay. KRAS mutation-specific forward primersand probes were:

for G12S mutation: Primer (SEQ ID NO: 26)5′-CTT GTG GTA GTT GGA GCA A-3′ Probe (SEQ ID NO: 27)5′-GCG CGT CCA GTG GCG TAG GC/3′C6/; for G12C mutation Primer(SEQ ID NO: 28) 5′-AAA CTT GTG GTA GTT GGA CCT T-3′ Probe(SEQ ID NO: 29) 5′-GCG CGT CCT GTG GCG TAG GC/3′C6/; for G12R mutationPrimer (SEQ ID NO: 30) 5′-TAT AAA CTT GTG GTA GTT GGA CCT C-3′ Probe(SEQ ID NO: 31) 5′-GCG CGT CCC GTG GCG TAG GC/3′C6/; for G12D mutationPrimer (SEQ ID NO: 32) 5′-ACT TGT GGT AGT TGG AGC TCA-3′ Probe(SEQ ID NO: 33) 5′-GCG CGT CCA TGG CGT AGG CA/3′C6/; for Gl2V mutationPrimer (SEQ ID NO: 34) 5′-ACT TGT GGT AGT TGG AGC TCT-3′ Probe(SEQ ID NO: 35) 5′-GCG CGT CCT TGG CGT AGG CA/3′C6/; for G12A mutationPrimer (SEQ ID NO: 36) 5′-AAC TTG TGG TAG TTG GAG ATG C-3′ Probe(SEQ ID NO: 37) 5′-GCG CGT CCC TGG CGT AGG CA/3′C6/; for G13D mutationPrimer (SEQ ID NO: 38) 5′-GGT AGT TGG AGC TGG TCA-3′ Probe(SEQ ID NO: 39) 5′-GCG CGT CCA CGT AGG CAA GA/3′C6/.

For all KRAS mutants, the reverse primer used is

(SEQ ID NO: 40) 5′-CTA TTG TTG GAT CAT ATT CGT C-3′.

QuARTS cycling conditions and reagent concentrations for KRAS were thesame as those in the methylation assays. Each plate contained standardsmade of engineered plasmids, positive and negative controls, and waterblanks, and was run in a LightCycler 480 (Roche). DNA strand number wasdetermined by comparing the C_(p) of the target gene to the standardcurve for that assay. The concentration of each mutation marker in 50microliters of KRAS was calculated based on the 500-fold dilution factorand an amplification efficiency of 1.95. This value was divided by theACTB concentration in the methylation assay and then multiplied by 100to determine the percent mutation.

Hemoglobin Assay.

To quantify hemoglobin in stool, the semi-automated HemoQuant test wasperformed on two buffered stool aliquots (each normalized to 16milligrams of stool) per patient, as described in Ahlquist, et al.(“HemoQuant, a new quantitative assay for fecal hemoglobin. Comparisonwith Hemoccult”. Ann Intern Med 101:297-302 (1984)). This test allowedassessment of the complementary value of fecal hemoglobin.

Data Analysis

Using the combination of sample processing methods described herein,comprising inhibitor removal and target capture purification, combinedwith the methylation and mutation markers described, the present studyof 678 samples achieved the following sensitivity levels: 63.8%sensitivity for adenoma detection and 85.3% sensitivity for colorectalcancer at a specificity level of 90%.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inpharmacology, biochemistry, medical science, or related fields areintended to be within the scope of the following claims.

We claim:
 1. A method of producing a filtrate from a sample, the methodcomprising: a) placing a sample to be filtered into a spin filter, saidspin filter comprising i) a hollow body (1); ii) a bottom end (2); andiii) an opening (3) at a top end of said hollow body (1) opposite thebottom end (2), wherein said hollow body (1) and said bottom end (2) areboth composed of the same porous filtering material; and b) centrifugingsaid spin filter, wherein during said centrifuging, a fraction of saidsample passes through said porous filtering material of said spin filterto produce a filtrate.
 2. The method of claim 1 wherein the porousfiltering material of said hollow body (1) and said bottom end (2) ofsaid spin filter is polyethylene.
 3. The method of claim 1 wherein theporous filtering material of said hollow body (1) and said bottom end(2) of said spin filter has a nominal pore size of 20 micrometers. 4.The method of claim 1 wherein the bottom end (2) of said spin filter hasa shape selected from the group consisting of a hemisphere, a disc, acone, or a portion of an ellipsoid.
 5. A method of claim 1, wherein saidspin filter is placed in a collection vessel adapted to receive the spinfilter, and wherein during said centrifuging, said filtrate is retainedin said collection vessel.